Weight loss frequently is observed in Alzheimer's disease (AD) patients prior to the onset of dementia, supportive of an underlying metabolic disorder. (Barrett-Connor et al., J Am Geriatr Soc. 44:1147-52 (1996); Bissoli et al., J Nutr Health Aging. 6:247-53 (2002)). Furthermore, lipid homeostasis (meaning the multi-layered regulatory networks of lipid metabolism, transport, and signal transduction) specifically, as exemplified in cell culture and animal models in addition to clinical studies with lipid-lowering agents, e.g., statins, can have an impact on amyloidogenic pathways. Such pathways lead to the generation of amyloid β (Aβ) peptide through proteolytic processing of the amyloid precursor protein (APP). (Stefan F. Lichtenthaler and Christian Haass, J Clin. Invest. 113:1384-1387 (2004); Puglielli et al., Nature Cell Biol. 3:905-912. (2001)). An important modulator of lipid homeostasis in non-adipose tissues is the pluripotent peptide leptin (Unger in Annu Rev Med. Vol. 53. 319-36 (2002).
In addition to deregulation of lipid metabolism in the CNS, the immune system has been implicated in the pathobiology of Alzheimer's disease. Amyloid plaques are decorated with proteins of the complement system, eicosanoids and cytokines, integral components of ongoing inflammatory processes that augment the harmful effects of Aβ (Emmerling et al., Biochim Biophys Acta 1502: 158-71 (2000)). Important regulators of the immune system include the cytokines and chemokines, secreted by leukocytes (B or T cells, normally scarce in the brain) or antigen presenting cells (APC) (microglia, perivascular macrophages, astrocytes in the brain). In AD brain, both pro-inflammatory cytokines and anti-inflammatory cytokines are expressed (Benveniste et al., Neurochem Int'l, 39: 381-91 (2001)). In addition to immune function, cytokines may directly affect the processing of APP (Blasko et al., FASEB J. 13: 63-68 (1999)). Leptin has very similar structural and functional characteristics to the cytokines (Heshka, J. T., and P. J. Jones, Life Sci. 69:987-1003 (2001)), sharing post-receptor pathways and participating in our immune response to pathogens and infections. Leptin deficiency is associated with impaired T cell immunity (Faggioni, R., K. R. Feingold, and C. Grunfeld. 2001. FASEB J. 15:2565-71 (2001)) and increased sensitivity to the lethal effects of bacterial endotoxin and TNF-a. Most importantly, these effects can be reversed with leptin administration, which attenuates inflammatory cytokine and neuroendocrine responses to infection (Xiao et al., Endocrinology 144: 4350-53 (2003)). Further, in critically ill septic patients, higher leptin levels are positively correlated with survival (Amalich et al., J. Infect. Dis. 180: 908-11 (1999)).
According to the present invention, the question of whether leptin and leptin signaling pathways are relevant to the pathology of a progressive brain disorder has been examined. The proposition is based on leptin's anti-amyloidogenic activity (Tezapsidis studies), leptin's ability to attenuate inflammation and leptin's ability to increase insulin sensitivity, a biological profile that could provide a multifaceted benefit to AD patients as a therapy and to the elderly as an intervention.
Leptin is a peptide hormone that controls adaptive metabolic mechanisms to energy availability leading to storage or mobilization of fat (Schwartz et al., Nature. 404: 661-71 (2000)). Adipocyte-derived leptin primarily exerts its central action through the arcuate nucleus neurons (an aggregation of neurons in the mediobasal hypothalamus); however, it can affect other populations, including hippocampal neurons and cells of the periphery (Shanley et al., Nat Neurosci. 5:299-300 (2002)). Ablation of leptin or of leptin signaling is sufficient to cause obesity as exemplified by leptin-deficient obese, hyperinsulinemic mice having the genotype ob/ob; diabetic mice with a mutation in the leptin receptor gene having the genotype db/db, which produce but are non-responsive to leptin; rats of the genotype fa/fa, which have the “fatty” obesity gene, which is a mutated leptin receptor; and in a few rare genetic cases (Schwartz et al., Nature. 404: 661-71 (2000)).
The leptin receptor (ObR), a member of the class I cytokine receptor superfamily (Lord, G. M., et al. Nature 394:897 (1998)) has at least six isoforms as a result of alternative splicing. As used herein the term “isoform” refers to a version of a protein that has the same function as another protein but that has some small differences in its sequence. All isoforms of ObR share an identical extracellular ligand-binding domain (Couce et al., Neuroendocrinology. 66:145-50 (1997)). Leptin's functional receptor (ObRb), the b isoform, is expressed not only in the hypothalamus, where it regulates energy homeostasis and neuroendocrine function, but also in other brain regions and in the periphery, including all cell types of innate and adaptive immunity (Lord, G. M., et al., Nature 394:897 (1998); Zhao, Y., R. et al., Biochem. Biophys. Res. Commun. 300: 247 (2003)); Zarkesh-Esfahani, H., G. et al., J. Immunol. 157: 4593 (2001) Caldefie-Chezet, F., A. et al., J. Leukocyte Biol. 69:414 (2001)). The full-length b isoform (ObRb) lacks intrinsic tyrosine kinase activity and is involved in several downstream signal transduction pathways.
Leptin binding to its functional receptor recruits Janus tyrosine kinases and activates the receptor, which then serves as a docking site for cytoplasmic adaptors such as STAT (Baumann, H., et al. Proc. Natl. Acad. Sci. USA 93:8374 1996)). According to the general model for JAK/STAT activation, STAT proteins initially are present in inactive forms in the cytoplasm. Following ligand stimulation and receptor dimerization, the JAK/STAT pathway is activated by activation of receptor-bound JAK kinases. These JAK kinases subsequently phosphorylate the receptor at tyrosine residues, which recruits STATs to the receptor. STATs then are phosphorylated to form phosphoSTATs, dimerized, and translocated to the nucleus, where the phosphoSTAT dimers bind to specific sequences in the promoter regions of their target genes, and stimulate the transcription of these genes (Schindler et al., Ann. Rev. Biochem. 64: 621-51 (1995)), including negative regulators, such as the suppressor of cytokine signaling 3 (Bjorbaek, C., K. et al. J. Biol. Chem. 274:30059 (1999)) and the protein tyrosine phosphatase 1B (Cheng, A. N. et al. Dev. Cell 2:497 (2002), Schwartz et al., Nature, 404:661-71 (2000), Louis A. Tartaglia, J. Biol. Chem. Minireview, 272:6093-6096 (March 1997)).
In addition to the JAK-2-STAT-3 pathway, other pathways also are involved in mediating leptin's effect in the brain and on the immune cells. For example, the mitogen-activated protein kinase (MAPK) pathways, the insulin receptor substrate 1 (IRS1) pathway, and the phosphatidylinositol 3′-kinase (PI3′K) pathway (Martin-Romero, C., V. Sanchez-Margalet. Cell. Immunol. 212:83 (2001)) also mediate leptin's action (Sanchez-Margalet, V., C. Martin-Romero, Cell. Immunol. 211:30 (2001)).
Leptin also may have a physiologic role as a liporegulatory hormone responsible for maintaining intracellular homeostasis in the face of wide variations in caloric intake (Unger R H. 2003. Annu Rev Physiol. 65:333-47). This is achieved by directly stimulating lipolysis, (meaning fat breakdown), and inhibiting lipogenesis (meaning fat synthesis) (Lee Y, et al., J. Biol Chem. 276(8):5629-35 (2001)). Leptin also can improve insulin resistance and hyperglycemia by a mechanism not completely understood (Toyoshima et al., Endocrinology 146: 4024-35 (2005)), despite insulin's ability to stimulate lipogenesis (Kersten, EMBO Reports 2(4): 282-286 (2001). This aspect of leptin's physiological role is important, because insulin and Aβ share a mechanism for their clearance, namely degradation by insulin degrading enzyme (IDE).
The levels of cholesterol and fatty acids in cells also are regulated tightly by a single family of transcription factors named Sterol Regulatory Element-Binding Proteins (SREBPs) which activate relevant target genes (Brown and Goldstein, Cell. 89:331-40 (1997)). SREBPs are transcription factors that regulate the expression of genes for both cholesterol and fatty acid synthesis. The inactive precursor form of SREBPs resides in cytoplasmic membranes. Intracellular lipid depletion triggers proteolytic cleavage of the SREBPs, allowing the amino terminus to enter the nucleus and activate the expression of enzymes, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), major biosynthetic enzymes for fatty acid synthesis. (Wilentz, Robb E. et al., Pediatric and Developmental Pathology, 3 (6): 525-531 (2000)).
In the central nervous system (CNS, meaning the brain and spinal cord), metabolic pathways involving lipids serve mainly to provide the building blocks for membranes, vitamins, second messengers and to modify proteins by acylation, because there are no main mechanisms for utilizing triglycerides/fatty acids as energy sources.
It is well documented that brain lipids are intricately involved in Amyloid β (Aβ)-related pathogenic pathways. The Aβ peptide is the major proteinaceous component of the amyloid plaques found in the brains of Alzheimer's disease (AD) patients and is regarded by many as the culprit of the disorder. The amount of extracellular Aβ accrued is critical for the pathobiology of AD and clearly depends on the antagonizing rates of its production/secretion and its clearance. It has been shown (Tezapsidis et al., FASEB J. 17:1322-1324 (2003)) that neurons depend on the interaction between Presenilin 1 (PS1) and Cytoplasmic-Linker Protein 170 (CLIP-170) to both generate Aβ and to take it up through the lipoprotein receptor related protein (LRP) pathway. Further to this requirement, formation of Aβ depends on the assembly of key proteins in lipid rafts (LRs) (Simons et al., Proc Natl Acad Sci USA. 95: 6460-4 (1998)). The term “lipid rafts” as used herein refers to membrane microdomains enriched in cholesterol, glycosphingolipids and glucosylphosphatidyl-inositol-(GPI)-tagged proteins implicated in signal transduction, protein trafficking and proteolysis. Within the LRs it is believed that Aβ's precursor, Amyloid Precursor Protein (APP), a type I membrane protein, is cleaved first by the protease β-secretase (BACE) to generate the C-terminal intermediate fragment of APP, CAPPβ, which remains imbedded in the membrane. The amino acid sequence of Aβ peptide showing its cleavage sites and membrane domain is shown in FIG. 1a. CAPPβ is subsequently cleaved at a site residing within the lipid bilayer by γ-secretase, a high molecular weight multi-protein complex containing presenilin, (PS1/PS2), nicastrin, PEN-2, and APH-1 or fragments thereof (De Strooper, Neuron. 38: 9-12 (2003)). Aβ finally is released outside the cell, where it can: a) start accumulating following oligomerization and exerting toxicity to neurons or b) be removed either by mechanisms of endocytosis (involving apolipoprotein-E (apoE) and LRP or Scavenger Receptors) or by degradation by extracellular proteases including insulin-degrading enzyme (IDE) and neprilysin (Farris et al., Proc Natl Acad Sci USA. 100:4162-4167 (2003)) (FIG. 1b).
Fatty acid and cholesterol availability and cellular composition modifies the transbilayer distribution of cholesterol and, consequently, overall membrane fluidity, function and localization of lipid rafts, a process which changes with aging (Wood et al., Neurobiol Aging. 23:685-694 (2002)). Therefore, it was hypothesized that leptin's lipolytic/antilipogenic activity could affect the composition of the LRs, affecting Aβ turnover.
The present invention demonstrates leptin's ability to modify the levels of Aβ both in vitro and in vivo. Leptin, similarly to methyl-β-cyclodextrin, reduces β-secretase activity in neuronal cells, possibly, but without being limited by theory, by altering the lipid composition of membrane LRs. This contrasts the results of treatments with cholesterol and etomoxir (an inhibitor of carnitine-palmitoyl transferase-1). Conversely, inhibitors of acetyl CoA carboxylase and fatty acid synthase mimicked leptin's action. Additionally, leptin was able to increase apoE-dependent Aβ uptake in vitro. Thus, according to the present invention, leptin can modulate indirectly bi-directional Aβ kinesis, reducing its levels extracellularly. Most strikingly, chronic administration of leptin to AD-transgenic animals reduced the brain Aβ load, illustrating its therapeutic potential.